Elevated MSH2 MSH3 expression interferes with DNA metabolism in vivo.

The Msh2-Msh3 mismatch repair (MMR) complex in Saccharomyces cerevisiae recognizes and directs repair of insertion/deletion loops (IDLs) up to ∼17 nucleotides. Msh2-Msh3 also recognizes and binds distinct looped and branched DNA structures with varying affinities, thereby contributing to genome stability outside post-replicative MMR through homologous recombination, double-strand break repair (DSBR) and the DNA damage response. In contrast, Msh2-Msh3 promotes genome instability through trinucleotide repeat (TNR) expansions, presumably by binding structures that form from single-stranded (ss) TNR sequences. We previously demonstrated that Msh2-Msh3 binding to 5' ssDNA flap structures interfered with Rad27 (Fen1 in humans)-mediated Okazaki fragment maturation (OFM) in vitro. Here we demonstrate that elevated Msh2-Msh3 levels interfere with DNA replication and base excision repair in vivo. Elevated Msh2-Msh3 also induced a cell cycle arrest that was dependent on RAD9 and ELG1 and led to PCNA modification. These phenotypes also required Msh2-Msh3 ATPase activity and downstream MMR proteins, indicating an active mechanism that is not simply a result of Msh2-Msh3 DNA-binding activity. This study provides new mechanistic details regarding how excess Msh2-Msh3 can disrupt DNA replication and repair and highlights the role of Msh2-Msh3 protein abundance in Msh2-Msh3-mediated genomic instability.

Robust Performance of SARS-CoV-2 Whole-Genome Sequencing from Wastewater with a Nonselective Virus Concentration Method.

The sequencing of human virus genomes from wastewater samples is an efficient method for tracking viral transmission and evolution at the community level. However, this requires the recovery of viral nucleic acids of high quality. We developed a reusable tangential-flow filtration system to concentrate and purify viruses from wastewater for genome sequencing. A pilot study was conducted with 94 wastewater samples from four local sewersheds, from which viral nucleic acids were extracted, and the whole genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was sequenced using the ARTIC V4.0 primers. Our method yielded a high probability (0.9) of recovering complete or near-complete SARS-CoV-2 genomes (>90% coverage at 10× depth) from wastewater when the COVID-19 incidence rate exceeded 33 cases per 100 000 people. The relative abundances of sequenced SARS-CoV-2 variants followed the trends observed from patient-derived samples. We also identified SARS-CoV-2 lineages in wastewater that were underrepresented or not present in the clinical whole-genome sequencing data. The developed tangential-flow filtration system can be easily adopted for the sequencing of other viruses in wastewater, particularly those at low concentrations.

Complex mutation profiles in mismatch repair and ribonucleotide reductase mutants reveal novel repair substrate specificity of MutS homolog (MSH) complexes.

Determining mutation signatures is standard for understanding the etiology of human tumors and informing cancer treatment. Multiple determinants of DNA replication fidelity prevent mutagenesis that leads to carcinogenesis, including the regulation of free deoxyribonucleoside triphosphate pools by ribonucleotide reductase and repair of replication errors by the mismatch repair system. We identified genetic interactions between rnr1 alleles that skew and/or elevate deoxyribonucleoside triphosphate levels and mismatch repair gene deletions. These defects indicate that the rnr1 alleles lead to increased mutation loads that are normally acted upon by mismatch repair. We then utilized a targeted deep-sequencing approach to determine mutational profiles associated with mismatch repair pathway defects. By combining rnr1 and msh mutations to alter and/or increase deoxyribonucleoside triphosphate levels and alter the mutational load, we uncovered previously unreported specificities of Msh2-Msh3 and Msh2-Msh6. Msh2-Msh3 is uniquely able to direct the repair of G/C single-base deletions in GC runs, while Msh2-Msh6 specifically directs the repair of substitutions that occur at G/C dinucleotides. We also identified broader sequence contexts that influence variant profiles in different genetic backgrounds. Finally, we observed that the mutation profiles in double mutants were not necessarily an additive relationship of mutation profiles in single mutants. Our results have implications for interpreting mutation signatures from human tumors, particularly when mismatch repair is defective.

A selection-based next generation sequencing approach to develop robust, genotype-specific mutation profiles in Saccharomyces cerevisiae.

Distinct mutation signatures arise from environmental exposures and/or from defects in metabolic pathways that promote genome stability. The presence of a particular mutation signature can therefore predict the underlying mechanism of mutagenesis. These insults to the genome often alter dNTP pools, which itself impacts replication fidelity. Therefore, the impact of altered dNTP pools should be considered when making mechanistic predictions based on mutation signatures. We developed a targeted deep-sequencing approach on the CAN1 gene in Saccharomyces cerevisiae to define information-rich mutational profiles associated with distinct rnr1 backgrounds. Mutations in the activity and selectivity sites of rnr1 lead to elevated and/or unbalanced dNTP levels, which compromises replication fidelity and increases mutation rates. The mutation spectra of rnr1Y285F and rnr1Y285A alleles were characterized previously; our analysis was consistent with this prior work but the sequencing depth achieved in our study allowed a significantly more robust and nuanced computational analysis of the variants observed, generating profiles that integrated information about mutation spectra, position effects, and sequence context. This approach revealed previously unidentified, genotype-specific mutation profiles in the presence of even modest changes in dNTP pools. Furthermore, we identified broader sequence contexts and nucleotide motifs that influenced variant profiles in different rnr1 backgrounds, which allowed specific mechanistic predictions about the impact of altered dNTP pools on replication fidelity.

Microscopic Communities: Interdisciplinary Exploration of Microbes in the Classroom.

Children are aware of microbes from a young age and are rightly encouraged to wash their hands to prevent illness. However, myriad microbes live in, on, and around us, most of which are benign or beneficial. Our goal was to teach elementary students about microbiota by leveraging familiar literacy practices, social studies themes, and the arts to advance students' knowledge and reasoning skills in science. With this perspective in mind, we developed and implemented an interdisciplinary unit targeted at second grade, in which students learned about microbes and microbial communities. Our goal was to further students' conceptual knowledge of the microbes that surround them by purposefully integrating microbial communities within the second grade curriculum. Throughout the unit, students engaged in hands-on, inquiry-based science experiences and used multimodal communication (through a combination of linguistic, visual, audio, gestural, and spatial modes): they sampled microbes from their own bodies and/or environments and applied their knowledge and imagination to create their own microbes through art and story-telling, generating a class microbial community-both literal and artistic. At the end of the unit, students demonstrated knowledge of microbes and of the diversity and ubiquity of microbial communities and habitats.

Tracking Expansions of Stable and Threshold Length Trinucleotide Repeat Tracts In Vivo and In Vitro Using Saccharomyces cerevisiae.

Trinucleotide repeat (TNR) tracts are inherently unstable during DNA replication, leading to repeat expansions and/or contractions. Expanded tracts are the cause of over 40 neurodegenerative and neuromuscular diseases. In this chapter, we focus on the (CAG)n and (CTG)n repeat sequences that, when expanded, lead to Huntington's disease (HD) and myotonic dystrophy type 1 (DM1), respectively, as well as a number of other neurodegenerative diseases. TNR tracts in most individuals are relatively small and stable in terms of length. However, TNR tracts become increasingly prone to expansion as tract length increases, eventually leading to very long tracts that disrupt coding (e.g. HD) or noncoding (e.g., DM1) regions of the genome. It is important to understand the early stages in TNR expansions, that is, the transition from small, stable lengths to susceptible threshold lengths. We describe PCR-based in vivo assays, using the model system Saccharomyces cerevisiae, to determine and characterize the dynamic behavior of TNR tracts in the stable and threshold ranges. We also describe a simple in vitro system to assess tract dynamics during 5' single-stranded DNA (ssDNA) flap processing and to assess the role of different DNA metabolism proteins in these dynamics. These assays can ultimately be used to determine factors that influence the early stages of TNR tract expansion.

Extracting and Measuring dNTP Pools in Saccharomyces cerevisiae.

Regulation of dNTP pools in an intracellular environment is not only vital for DNA replication but also plays a major role in maintaining genomic stability. Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in dNTP synthesis and altered regulation of RNR leads to imbalanced dNTP pools. Increased dNTP levels are mutagenic and have the potential to interfere with pathways that are involved in DNA replication, repair and DNA damage control. However, the mechanisms through which altered dNTP pools affect these pathways are poorly understood. Nonetheless, altered dNTP pools have been identified in a number of cellular contexts, including cancer. In order to interpret and analyze the effects of altered dNTP pools, we need quantitative information about dNTP pools in different genetic and environmental contexts in vivo. Here we describe a high-throughput fluorescence-based assay that uses a qPCR-based approach to quantify dNTP levels for use with Saccharomyces cerevisiae extracts.

Distinct roles of XPF-ERCC1 and Rad1-Rad10-Saw1 in replication-coupled and uncoupled inter-strand crosslink repair.

Yeast Rad1-Rad10 (XPF-ERCC1 in mammals) incises UV, oxidation, and cross-linking agent-induced DNA lesions, and contributes to multiple DNA repair pathways. To determine how Rad1-Rad10 catalyzes inter-strand crosslink repair (ICLR), we examined sensitivity to ICLs from yeast deleted for SAW1 and SLX4, which encode proteins that interact physically with Rad1-Rad10 and bind stalled replication forks. Saw1, Slx1, and Slx4 are critical for replication-coupled ICLR in mus81 deficient cells. Two rad1 mutations that disrupt interactions between Rpa1 and Rad1-Rad10 selectively disable non-nucleotide excision repair (NER) function, but retain UV lesion repair. Mutations in the analogous region of XPF also compromised XPF interactions with Rpa1 and Slx4, and are proficient in NER but deficient in ICLR and direct repeat recombination. We propose that Rad1-Rad10 makes distinct contributions to ICLR depending on cell cycle phase: in G1, Rad1-Rad10 removes ICL via NER, whereas in S/G2, Rad1-Rad10 facilitates NER-independent replication-coupled ICLR.

Coordination of Rad1-Rad10 interactions with Msh2-Msh3, Saw1 and RPA is essential for functional 3' non-homologous tail removal.

Double strand DNA break repair (DSBR) comprises multiple pathways. A subset of DSBR pathways, including single strand annealing, involve intermediates with 3' non-homologous tails that must be removed to complete repair. In Saccharomyces cerevisiae, Rad1-Rad10 is the structure-specific endonuclease that cleaves the tails in 3' non-homologous tail removal (3' NHTR). Rad1-Rad10 is also an essential component of the nucleotide excision repair (NER) pathway. In both cases, Rad1-Rad10 requires protein partners for recruitment to the relevant DNA intermediate. Msh2-Msh3 and Saw1 recruit Rad1-Rad10 in 3' NHTR; Rad14 recruits Rad1-Rad10 in NER. We created two rad1 separation-of-function alleles, rad1R203A,K205A and rad1R218A; both are defective in 3' NHTR but functional in NER. In vitro, rad1R203A,K205A was impaired at multiple steps in 3' NHTR. The rad1R218A in vivo phenotype resembles that of msh2- or msh3-deleted cells; recruitment of rad1R218A-Rad10 to recombination intermediates is defective. Interactions among rad1R218A-Rad10 and Msh2-Msh3 and Saw1 are altered and rad1R218A-Rad10 interactions with RPA are compromised. We propose a model in which Rad1-Rad10 is recruited and positioned at the recombination intermediate through interactions, between Saw1 and DNA, Rad1-Rad10 and Msh2-Msh3, Saw1 and Msh2-Msh3 and Rad1-Rad10 and RPA. When any of these interactions is altered, 3' NHTR is impaired.

Measuring Dynamic Behavior of Trinucleotide Repeat Tracts In Vivo in Saccharomyces cerevisiae.

Trinucleotide repeat (TNR) tracts are inherently unstable during DNA replication, leading to repeat expansions and/or contractions. Expanded tracts are the cause of over 40 neurodegenerative and neuromuscular diseases. In this chapter, we focus on the (CNG)n repeat sequences that, when expanded, lead to Huntington's disease (HD), myotonic dystrophy type 1 (DM1), and a number of other neurodegenerative diseases. We describe a series of in vivo assays, using the model system Saccharomyces cerevisiae, to determine and characterize the dynamic behavior of TNR tracts that are in the early stages of expansion, i.e., the so-called threshold range. Through a series of time courses and PCR-based assays, dynamic changes in tract length can be observed as a function of time. These assays can ultimately be used to determine how genetic factors influence the process of tract expansion in these early stages.

mlh3 mutations in baker's yeast alter meiotic recombination outcomes by increasing noncrossover events genome-wide.

Mlh1-Mlh3 is an endonuclease hypothesized to act in meiosis to resolve double Holliday junctions into crossovers. It also plays a minor role in eukaryotic DNA mismatch repair (MMR). To understand how Mlh1-Mlh3 functions in both meiosis and MMR, we analyzed in baker's yeast 60 new mlh3 alleles. Five alleles specifically disrupted MMR, whereas one (mlh3-32) specifically disrupted meiotic crossing over. Mlh1-mlh3 representatives for each class were purified and characterized. Both Mlh1-mlh3-32 (MMR+, crossover-) and Mlh1-mlh3-45 (MMR-, crossover+) displayed wild-type endonuclease activities in vitro. Msh2-Msh3, an MSH complex that acts with Mlh1-Mlh3 in MMR, stimulated the endonuclease activity of Mlh1-mlh3-32 but not Mlh1-mlh3-45, suggesting that Mlh1-mlh3-45 is defective in MSH interactions. Whole genome recombination maps were constructed for wild-type and MMR+ crossover-, MMR- crossover+, endonuclease defective and null mlh3 mutants in an S288c/YJM789 hybrid background. Compared to wild-type, all of the mlh3 mutants showed increases in the number of noncrossover events, consistent with recombination intermediates being resolved through alternative recombination pathways. Our observations provide a structure-function map for Mlh3 that reveals the importance of protein-protein interactions in regulating Mlh1-Mlh3's enzymatic activity. They also illustrate how defective meiotic components can alter the fate of meiotic recombination intermediates, providing new insights for how meiotic recombination pathways are regulated.

The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans.

Crossing over between homologs is initiated in meiotic prophase by the formation of DNA double-strand breaks that occur throughout the genome. In the major interference-responsive crossover pathway in baker's yeast, these breaks are resected to form 3' single-strand tails that participate in a homology search, ultimately forming double Holliday junctions (dHJs) that primarily include both homologs. These dHJs are resolved by endonuclease activity to form exclusively crossovers, which are critical for proper homolog segregation in Meiosis I. Recent genetic, biochemical, and molecular studies in yeast are consistent with the hypothesis of Mlh1-Mlh3 DNA mismatch repair complex acting as the major endonuclease activity that resolves dHJs into crossovers. However, the mechanism by which the Mlh1-Mlh3 endonuclease is activated is unknown. Here, we provide evidence that Mlh1-Mlh3 does not behave like a structure-specific endonuclease but forms polymers required to generate nicks in DNA. This conclusion is supported by DNA binding studies performed with different-sized substrates that contain or lack polymerization barriers and endonuclease assays performed with varying ratios of endonuclease-deficient and endonuclease-proficient Mlh1-Mlh3. In addition, Mlh1-Mlh3 can generate religatable double-strand breaks and form an active nucleoprotein complex that can nick DNA substrates in trans. Together these observations argue that Mlh1-Mlh3 may not act like a canonical, RuvC-like Holliday junction resolvase and support a novel model in which Mlh1-Mlh3 is loaded onto DNA to form an activated polymer that cleaves DNA.

Large-scale production of recombinant Saw1 in Escherichia coli.

Saccharomyces cerevisiae Saw1 is an essential gene in single-strand annealing - the DNA repair pathway that repairs double-strand breaks when they occur between homologous repeats. Saw1 interacts with the structure-specific nuclease Rad1-Rad10 and this results in the recruitment of this nuclease to 3' non-homologous DNA tailed recombination intermediates. Saw1 is unstable in the absence of the Rad1-Rad10 nuclease and, hence, it has been difficult to study its specific function in vitro. In the present work, we present the combination of dynamic light scattering and differential scanning fluorimetry techniques to optimize the stability and homogeneity of recombinant Saw1. The protein expression and purification conditions identified in this study allow for higher recovery of soluble Saw1 and enable the biochemical characterization of the protein.

Dynamic DNA binding licenses a repair factor to bypass roadblocks in search of DNA lesions.

DNA-binding proteins search for specific targets via facilitated diffusion along a crowded genome. However, little is known about how crowded DNA modulates facilitated diffusion and target recognition. Here we use DNA curtains and single-molecule fluorescence imaging to investigate how Msh2-Msh3, a eukaryotic mismatch repair complex, navigates on crowded DNA. Msh2-Msh3 hops over nucleosomes and other protein roadblocks, but maintains sufficient contact with DNA to recognize a single lesion. In contrast, Msh2-Msh6 slides without hopping and is largely blocked by protein roadblocks. Remarkably, the Msh3-specific mispair-binding domain (MBD) licences a chimeric Msh2-Msh6(3MBD) to bypass nucleosomes. Our studies contrast how Msh2-Msh3 and Msh2-Msh6 navigate on a crowded genome and suggest how Msh2-Msh3 locates DNA lesions outside of replication-coupled repair. These results also provide insights into how DNA repair factors search for DNA lesions in the context of chromatin.

MSH3 Promotes Dynamic Behavior of Trinucleotide Repeat Tracts In Vivo.

Trinucleotide repeat (TNR) expansions are the underlying cause of more than 40 neurodegenerative and neuromuscular diseases, including myotonic dystrophy and Huntington's disease, yet the pathway to expansion remains poorly understood. An important step in expansion is the shift from a stable TNR sequence to an unstable, expanding tract, which is thought to occur once a TNR attains a threshold length. Modeling of human data has indicated that TNR tracts are increasingly likely to expand as they increase in size and to do so in increments that are smaller than the repeat itself, but this has not been tested experimentally. Genetic work has implicated the mismatch repair factor MSH3 in promoting expansions. Using Saccharomyces cerevisiae as a model for CAG and CTG tract dynamics, we examined individual threshold-length TNR tracts in vivo over time in MSH3 and msh3Δ backgrounds. We demonstrate, for the first time, that these TNR tracts are highly dynamic. Furthermore, we establish that once such a tract has expanded by even a few repeat units, it is significantly more likely to expand again. Finally, we show that threshold- length TNR sequences readily accumulate net incremental expansions over time through a series of small expansion and contraction events. Importantly, the tracts were substantially stabilized in the msh3Δ background, with a bias toward contractions, indicating that Msh2-Msh3 plays an important role in shifting the expansion-contraction equilibrium toward expansion in the early stages of TNR tract expansion.

ATP binding and hydrolysis by Saccharomyces cerevisiae Msh2-Msh3 are differentially modulated by mismatch and double-strand break repair DNA substrates.

In Saccharomyces cerevisiae, Msh2-Msh3-mediated mismatch repair (MMR) recognizes and targets insertion/deletion loops for repair. Msh2-Msh3 is also required for 3' non-homologous tail removal (3'NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, we recently demonstrated that the two pathways have distinct requirements with respect to Msh2-Msh3 activities. We identified a set of aromatic residues in the nucleotide binding pocket (FLY motif) of Msh3 that, when mutated, disrupted MMR, but left 3'NHTR largely intact. One of these mutations, msh3Y942A, was predicted to disrupt the nucleotide sandwich and allow altered positioning of ATP within the pocket. To develop a mechanistic understanding of the differential requirements for ATP binding and/or hydrolysis in the two pathways, we characterized Msh2-Msh3 and Msh2-msh3Y942A ATP binding and hydrolysis activities in the presence of MMR and 3'NHTR DNA substrates. We observed distinct, substrate-dependent ATP hydrolysis and nucleotide turnover by Msh2-Msh3, indicating that the MMR and 3'NHTR DNA substrates differentially modify the ATP binding/hydrolysis activities of Msh2-Msh3. Msh2-msh3Y942A retained the ability to bind DNA and ATP but exhibited altered ATP hydrolysis and nucleotide turnover. We propose that both ATP and structure-specific repair substrates cooperate to direct Msh2-Msh3-mediated repair and suggest an explanation for the msh3Y942A separation-of-function phenotype.

Distinct requirements within the Msh3 nucleotide binding pocket for mismatch and double-strand break repair.

In Saccharomyces cerevisiae, repair of insertion/deletion loops is carried out by Msh2-Msh3-mediated mismatch repair (MMR). Msh2-Msh3 is also required for 3' non-homologous tail removal (3' NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, the kinetics of the two processes appear different; MMR is likely rapid in order to coordinate with the replication fork, whereas 3' NHTR has been shown to be a slower process. To understand the molecular requirements in both repair pathways, we performed an in vivo analysis of well-conserved residues in Msh3 that are hypothesized to be required for MMR and/or 3' NHTR. These residues are predicted to be involved in either communication between the DNA-binding and ATPase domains within the complex or nucleotide binding and/or exchange within Msh2-Msh3. We identified a set of aromatic residues within the FLY motif of the predicted Msh3 nucleotide binding pocket that are essential for Msh2-Msh3-mediated MMR but are largely dispensable for 3' NHTR. In contrast, mutations in other regions gave similar phenotypes in both assays. Based on these results, we suggest that the two pathways have distinct requirements with respect to the position of the bound ATP within Msh3. We propose that the differences are related, at least in part, to the kinetics of each pathway. Proper binding and positioning of ATP is required to induce rapid conformational changes at the replication fork, but is less important when more time is available for repair, as in 3' NHTR.

Role of Saw1 in Rad1/Rad10 complex assembly at recombination intermediates in budding yeast.

The Saccharomyces cerevisiae Rad1/Rad10 complex is a multifunctional, structure-specific endonuclease that processes UV-induced DNA lesions, recombination intermediates, and inter-strand DNA crosslinks. However, we do not know how Rad1/Rad10 recognizes these structurally distinct target molecules or how it is incorporated into the protein complexes capable of incising divergent substrates. Here, we have determined the order and hierarchy of assembly of the Rad1/Rad10 complex, Saw1, Slx4, and Msh2/Msh3 complex at a 3' tailed recombination intermediate. We found that Saw1 is a structure-specific DNA binding protein with high affinity for splayed arm and 3'-flap DNAs. By physical interaction, Saw1 facilitates targeting of Rad1 at 3' tailed substrates in vivo and in vitro, and enhances 3' tail cleavage by Rad1/Rad10 in a purified system in vitro. Our results allow us to formulate a model of Rad1/Rad10/Saw1 nuclease complex assembly and 3' tail removal in recombination.

Msh2-Msh3 interferes with Okazaki fragment processing to promote trinucleotide repeat expansions.

Trinucleotide repeat (TNR) expansions are the underlying cause of more than 40 neurodegenerative and neuromuscular diseases, including myotonic dystrophy and Huntington's disease. Although genetic evidence points to errors in DNA replication and/or repair as the cause of these diseases, clear molecular mechanisms have not been described. Here, we focused on the role of the mismatch repair complex Msh2-Msh3 in promoting TNR expansions. We demonstrate that Msh2-Msh3 promotes CTG and CAG repeat expansions in vivo in Saccharomyces cerevisiae. Furthermore, we provide biochemical evidence that Msh2-Msh3 directly interferes with normal Okazaki fragment processing by flap endonuclease1 (Rad27) and DNA ligase I (Cdc9) in the presence of TNR sequences, thereby producing small, incremental expansion events. We believe that this is the first mechanistic evidence showing the interplay of replication and repair proteins in the expansion of sequences during lagging-strand DNA replication.

Multiple factors insulate Msh2-Msh6 mismatch repair activity from defects in Msh2 domain I.

DNA mismatch repair (MMR) is a highly conserved mutation avoidance mechanism that corrects DNA polymerase misincorporation errors. In initial steps in MMR, Msh2-Msh6 binds mispairs and small insertion/deletion loops, and Msh2-Msh3 binds larger insertion/deletion loops. The msh2Δ1 mutation, which deletes the conserved DNA-binding domain I of Msh2, does not dramatically affect Msh2-Msh6-dependent repair. In contrast, msh2Δ1 mutants show strong defects in Msh2-Msh3 functions. Interestingly, several mutations identified in patients with hereditary non-polyposis colorectal cancer map to domain I of Msh2; none have been found in MSH3. To understand the role of Msh2 domain I in MMR, we examined the consequences of combining the msh2Δ1 mutation with mutations in two distinct regions of MSH6 and those that increase cellular mutational load (pol3-01 and rad27). These experiments reveal msh2Δ1-specific phenotypes in Msh2-Msh6 repair, with significant effects on mutation rates. In vitro assays demonstrate that msh2Δ1-Msh6 DNA binding is less specific for DNA mismatches and produces an altered footprint on a mismatch DNA substrate. Together, these results provide evidence that, in vivo, multiple factors insulate MMR from defects in domain I of Msh2 and provide insights into how mutations in Msh2 domain I may cause hereditary non-polyposis colorectal cancer.